RESUMEN
Point mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease and augment LRRK2's kinase activity. However, cellular pathways that endogenously enhance LRRK2 kinase function have not been identified. While overexpressed Rab29 draws LRRK2 to Golgi membranes to increase LRRK2 kinase activity, there is little evidence that endogenous Rab29 performs this function under physiological conditions. Here, we identify Rab38 as a novel physiologic regulator of LRRK2 in melanocytes. In mouse melanocytes, which express high levels of Rab38, Rab32, and Rab29, knockdown (or CRISPR knockout) of Rab38, but not Rab32 or Rab29, decreases phosphorylation of multiple LRRK2 substrates, including Rab10 and Rab12, by both endogenous LRRK2 and exogenous Parkinson's disease-mutant LRRK2. In B16-F10 mouse melanoma cells, Rab38 drives LRRK2 membrane association and overexpressed kinase-active LRRK2 shows striking pericentriolar recruitment, which is dependent on the presence of endogenous Rab38 but not Rab32 or Rab29. Consistently, knockdown or mutation of BLOC-3, the guanine nucleotide exchange factor for Rab38 and Rab32, inhibits Rab38's regulation of LRRK2. Deletion or mutation of LRRK2's Rab38-binding site in the N-terminal armadillo domain decreases LRRK2 membrane association, pericentriolar recruitment, and ability to phosphorylate Rab10. In sum, our data identify Rab38 as a physiologic regulator of LRRK2 function and lend support to a model in which LRRK2 plays a central role in Rab GTPase coordination of vesicular trafficking.
Asunto(s)
Membranas Intracelulares , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Melanocitos , Proteínas de Unión al GTP rab , Animales , Ratones , Aparato de Golgi/enzimología , Aparato de Golgi/genética , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Melanocitos/metabolismo , Mutación , Enfermedad de Parkinson/metabolismo , Fosforilación , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Células HEK293 , Humanos , Expresión Génica , Dominios Proteicos , Unión Proteica , Membranas Intracelulares/metabolismoRESUMEN
Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a disease that kills an estimated 200,000 people annually. Previously, we discovered an antimicrobial pathway dependent on Rab32 and BLOC-3 (BRAM) that is critical to kill S. Typhi in murine macrophages. The BLOC-3 complex is comprised of the two sub-units HPS1 and HPS4 and exhibits guanine-nucleotide exchange factor (GEF) activity to Rab32. In melanocytes, Rab9 has been shown to interact with HPS4 and RUTBC1, a Rab32 GTPase activating (GAP) protein, and regulate the Rab32-mediated melanosome biogenesis. Intriguingly, Rab9-deficient melanocytes exhibit hypopigmentation, a similar phenotype to Rab32 or BLOC-3 deficient melanocytes. Additionally, VPS9-ankyrin-repeat-protein (VARP) has been shown to regulate melanocytic enzyme trafficking into the melanosomes through interaction with Rab32. Although Rab32, Rab9 and VARP are a part of melanogenesis in melanocytes, whether Rab9 and VARP are required for the BRAM mediated killing in macrophages is currently unknown. Here we showed that HPS4 is recruited to the Salmonella-containing vacuoles (SCV) and over-expression of BLOC-3 significantly increased Rab32-positive bacteria vacuoles. We found that SCV acquire Rab9, however over-expressing Rab9 did not change HPS4 localization on bacteria vacuoles. Importantly, we used shRNA to knock-down Rab9 and VARP in macrophages and showed that these proteins are dispensable for Rab32 recruitment to the SCV. Furthermore, we assessed the survival of S. Typhimurium in macrophages deficient for Rab9 or VARP and demonstrated that these proteins are not essential for BRAM pathway-dependent killing.
Asunto(s)
Melanosomas , Proteínas de Unión al GTP rab , Animales , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Melanosomas/metabolismo , Ratones , Salmonella typhi/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Albinism, which is commonly inherited as an autosomal recessive trait, is characterized by a reduction or absence of melanin in the eyes, skin, and hair. To date, more than 20 causal genes for albinism have been identified; thus, the accurate diagnosis of albinism requires next-generation sequencing (NGS). In this study, we analyzed 46 patients who tested negative for oculocutaneous albinism (OCA)1-4 and Hermansky-Pudlak syndrome (HPS)1 based on conventional analysis, in addition to 28 new Japanese patients, using NGS-based targeted resequencing. We identified a genetic background for albinism in 18 of the 46 patients (39%), who were previously tested negative according to the conventional analysis. In addition, we unveiled a genetic predisposition toward albinism in 23 of the 28 new patients (82%). We identified six patients with rare subtypes of albinism, including HPS3, HPS4, and HPS6, and found 12 novel pathological mutations in albinism-related genes. Furthermore, most patients who were not diagnosed with albinism by the NGS analysis showed mild manifestations of albinism without apparent eye symptoms and harbored only one heterozygous mutation, occasionally in combination with skin-color associated gene variants.
Asunto(s)
Albinismo/diagnóstico , Albinismo/genética , Pueblo Asiatico/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Adulto , Secuencia de Bases , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Japón , Masculino , Persona de Mediana Edad , Mutación/genética , Fenotipo , Sitios de Empalme de ARN/genética , Adulto JovenRESUMEN
Lysosome-related organelles (LROs) comprise a diverse group of cell type-specific, membrane-bound subcellular organelles that derive at least in part from the endolysosomal system but that have unique contents, morphologies and functions to support specific physiological roles. They include: melanosomes that provide pigment to our eyes and skin; alpha and dense granules in platelets, and lytic granules in cytotoxic T cells and natural killer cells, which release effectors to regulate hemostasis and immunity; and distinct classes of lamellar bodies in lung epithelial cells and keratinocytes that support lung plasticity and skin lubrication. The formation, maturation and/or secretion of subsets of LROs are dysfunctional or entirely absent in a number of hereditary syndromic disorders, including in particular the Hermansky-Pudlak syndromes. This review provides a comprehensive overview of LROs in humans and model organisms and presents our current understanding of how the products of genes that are defective in heritable diseases impact their formation, motility and ultimate secretion.
Asunto(s)
Síndrome de Hermanski-Pudlak/metabolismo , Lisosomas/metabolismo , Melanosomas/metabolismo , Cuerpos de Weibel-Palade/metabolismo , Animales , Síndrome de Hermanski-Pudlak/patología , Humanos , Lisosomas/ultraestructura , Melanosomas/ultraestructura , Cuerpos de Weibel-Palade/ultraestructuraRESUMEN
HPS4 biogenesis of lysosome-related organelles complex 3 subunit 2 (HPS4) is one of the genes whose mutations have been associated with Hermansky-Pudlak syndrome (HPS), characterized by ocular albinism and susceptibility to bleeding because of defects in the biogenesis of lysosome-related organelles such as melanosomes. HPS4 protein forms a BLOC-3 complex with HPS1, another HPS gene product, and the complex has been proposed to function as a guanine nucleotide exchange factor (GEF) for RAB32, a member of the Rab small GTPase family (Rab32), and Rab38 (Rab32/38-GEF) and also as a Rab9 effector. Although both Rab32/38 and Rab9 have been shown previously to be involved in melanogenesis in mammalian epidermal melanocytes, the functional relationships of these small GTPases with BLOC-3 remain unknown. In this study, we used site-directed mutagenesis to generate HPS4 mutants that specifically lack either Rab32/38-GEF activity or Rab9-binding activity and investigated their involvement in melanogenesis of melan-le cells (an HPS4-deficient melanocyte cell line derived from light ear mice). Melan-le cells exhibit a clear hypopigmentation phenotype, i.e. reduced expression and abnormal distribution of tyrosinase and reduced melanin content. Although re-expression of WT HPS4 completely rescued this phenotype, the Rab32/38-GEF activity-deficient HPS4 mutant failed to restore melanin content and tyrosinase trafficking in these cells. Unexpectedly, as WT HPS4, the Rab9 binding-deficient HPS4 mutant completely rescued the phenotype. These results indicate that activation of Rab32/38 by HPS4 (or BLOC-3) is essential for melanogenesis of cultured melanocytes and that Rab9 likely regulates melanogenesis independently of HPS4.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/fisiología , Melaninas/biosíntesis , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular Transformada , Chlorocebus aethiops , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/química , Ratones , Mutagénesis Sitio-Dirigida , Unión ProteicaRESUMEN
Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.
Asunto(s)
Endocitosis , Endosomas/metabolismo , Melanosomas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Lisosomas/metabolismo , Melanocitos/metabolismo , Ratones , Modelos Biológicos , Pigmentación , Transporte de Proteínas , ARN Interferente Pequeño/metabolismoRESUMEN
The eye has pigmented cells of two different embryonic origins and therefore it is a good model for studying melanosome biogenesis and melanin production/deposition. Pale ear mice bear a mutation in the Hermansky-Pudlak syndrome type 1 (HPS-1) gene and exhibit abnormal eye pigmentation. Here, we reported the delayed and reduced pigmentation in eyes of pale ear mice in early postnatal stages and adulthood. Tyrosinase assay and L-3,4-dihydroxyphenylalanine (L-DOPA) gel staining assay revealed that tyrosinase activity in eyes of pale ear mutants was greatly reduced in early postnatal stages and increased gradually after postnatal day 7 (P7). Further histological examination revealed that hypopigmentation in the retinal pigment epithelium (RPE) and pigment epithelium of the iris and ciliary body, which are derived from the optic cup, was more severe than that in neural crest-derived tissues. In addition, macromelanosomes were exclusively present in neural crest-derived melanocytes of pale ear adults, but absent at early postnatal stages. Taken together, the mutation in the HPS-1 gene could cause two distinct phenotypes in pigmented cells of different embryonic origins. Besides, an increased accumulation of lipofuscin in RPE was also observed.